The HIV-1 envelope protein (Env) is a class 1 membrane fusion machine that mediates virus entry into cells. HIV-1 Env consists of a trimer of gp120/gp41 heterodimers. Interaction with the CD4 receptor causes structural rearrangements in gp120, which lead to formation of a coreceptor-binding site. Subsequent interactions with the coreceptor trigger additional Env refolding, with gp41 rearranging into a stable six-helix bundle that is believed to drive fusion between viral and cellular membranes. Recent progress in the structural understanding have provided static images of the pre-fusion conformation of the HIV-1 Env trimer and post-fusion conformation of gp41. However, direct visualization of the conformational transitions, characterization of structural intermediates that lead to membrane fusion have been lacking. To provide insights into the dynamics of the native trimer, we have established single molecule fluorescence resonance energy transfer (smFRET) imaging to measure the conformational changes of individual Env molecule in the context of a native trimer on the surface of intact virions. Our studies revealed that the unliganded HIV-1 Env is intrinsically dynamic, transitioning between three distinct prefusion conformations, whose relative occupancies were remodeled by receptor CD4 and antibody binding. Our analysis also directly reveals molecular and temporal events in gp120 that underlie the two-step activation of HIV-1 Env by CD4 and coreceptor through a necessary structural intermediate. Here we propose to concentrate on the next important goal, to directly visualize the conformational events within gp41 that lead to mixing between viral and cellular membranes. Towards this end, we have established suppressor tRNA technologies to introduce unnatural amino acids in mammalian cells. The introduction of a single fluorophore in gp41 in combination with an existing one in gp120 will monitor how CD4 primes gp41 and binding of co-receptor disrupts the association between gp41 and gp120. The insertion of two fluorophores before and after switch regions within HR1 and HR2 regions of gp41 will reveal how individual conformational transitions within the Env trimer lead to fusion peptide exposure and membrane fusion. Because our methods allows insights into the conformational state of native HIV-1 Env trimer on the surface of complete virions, we are also uniquely positioned to evaluate to what extent soluble or precursor constructs currently used to structurally characterize the HIV-1 Env trimer display features of the native Env. An increased understanding of the conformational events underlying HIV-1 Env activation will inform antiviral therapies and vaccine design.